Jak3 is a protein kinase required for signaling through cytokine receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Previous studies have shown that mutations in Jak3 result in a severe combined immunodeficiency syndrome (SCID) in humans, indicating that Jak3 plays an absolutely essential role in the function of the immune system. In addition, pharmacological inhibitors of Jak3 are currently being developed for use as immunosuppressants. Nonetheless, the precise role of Jak3 in T cell activation, differentiation, and homeostasis is currently uncertain, as are the consequences of long-term inhibition of Jak3 activity. We have developed an animal model using Jak3-deficient mice that will allow us to address the role of Jak3- dependent cytokine signaling in normal cells and in intact animals. Our recent data, based on findings from gene expression profiling and cytokine analysis experiments, indicate that the CD4+ T cells present in Jak3-/- mice closely resemble regulatory T cells of the Treg-1 (Tr1) phenotype. These cells are non- proliferative in vitro, and secrete large amounts of IFN-g, IL-10, and TGF-? following TCR stimulation, but no IL-2 or IL-4. In addition, they have high constitutive levels of the inhibitory surface receptor, PD-1. Preliminary data also suggest that these Jak3-/- T cells can suppress proliferative responses of wild type T cells in vitro. Our hypothesis is that Jak3-dependent cytokine signals are critical for the differentiation of activated T cells into functional, conventional effector T cells, and that in the absence of these signals, activated T cells differentiate into Tr1 regulatory T cells. We also hypothesize that the "activation" of Jak3-/- CD4+ T cells results from the homeostatic proliferation of these cells in the lymphophenic environment of the Jak3-/- host. To test these hypotheses, we propose to determine whether Jak3 -/- CD4+ T cells function as regulatory T cells, and the role(s) of IL-10, TGF-b, and PD-1 in this function. Second, we will examine the differentiation pathway of naive Jak3-/- CD4+ T cells following antigen stimulation in vitro and in vivo, and following homeostatic expansion in a lymphopenic wild type host. Finally, we will determine the role of Stat5 as a downstream mediator of Jak-3 dependent cytokine signals in regulating effector T cell versus regulatory T cell differentiation pathways. [unreadable] [unreadable] [unreadable]